Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS–PCR

Chiapparino, Elena; Lee, D; Donini, Paolo

doi:10.1139/g03-130

Cited by 57 publications

(30 citation statements)

References 19 publications

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“…However, with six out of the above seven allele specific primers, a homoeologue specific band (large fragment) was observed in 37 to 42 genotypes. The above observation is in conformity with earlier studies conducted in barley (Chiapparino et al 2004;Bundock et al 2005), where the failure of genome specific amplification was attributed to the competition among one of the locus specific primers and allele specific primers used during the PCR reaction.…”

Section: Development Of Pcr Based Assay For Snp Genotypingsupporting

confidence: 92%

“…Seven (7) putative SNPs validated through direct sequencing of PCR products were used to develop allele specific primers using Primer3 (for primer details, see Table 3) following the strategy described elsewhere (Jeong et al 2004;Chiapparino et al 2004). The allele specific primers varied in length from 18 to 30 bases and the annealing temperatures ranged from 58.3°C to 64.2°C (Table 3).…”

Section: Validation Of Snps and Development Of Pcr Based Assaymentioning

confidence: 99%

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EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

Rustgi¹,

Bandopadhyay²,

Balyan³

et al. 2009

CZECH J GENET PLANT

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Abstract:The present study involves discovery, validation and use of single-nucleotide polymorphisms (SNPs) in bread wheat utilizing 48 EST-contigs (individual contigs having 20-89 ESTs, derived from 2 to 11 different genotypes). In order to avoid a problem due to homoeologous relationships, the ESTs in each contig were classified into 175 sub-contigs (3.7 sub-contigs/EST-contig) using characteristic homoeologue sequence variants (HSVs), which had a density of 1 HSV every 136.7 bp. In silico analysis of sub-contigs led to the discovery of 230 candidate EST-SNPs with a density of 1SNP/273.9 bp. Locus specific primers (each primer pair flanking 1-18 SNPs) were designed utilizing one sub-contig each from 42 EST-contigs that contained SNPs, the remaining 6 contigs having no SNPs. To provide locus specificity to the PCR products, each primer was tagged with an HSV at its 3' end. Only 10 primer pairs, which gave each a characteristic solitary band, were utilized to validate EST-SNPs over 30 diverse bread wheat genotypes; 7 SNPs were validated through resequencing the PCR products. Allele specific primers were designed and utilized for genotyping of 50 diverse bread wheat accessions (including 30 bread wheat genotypes previously used for validation of SNPs), with an aim to test their utility in genotyping and map construction. The allele specific primers allowed the classification of 50 genotypes in two alternative allele groups for each SNP as expected, thus suggesting their utility for genotyping. Of the above 7 validated SNPs, 4 belonged to a solitary locus (PKS37); 7 haplotypes were available at this locus. Altogether, the results suggested that EST-SNPs constitute an important source of molecular markers for studies on wheat genomics.

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Section: Development Of Pcr Based Assay For Snp Genotypingsupporting

confidence: 92%

Section: Validation Of Snps and Development Of Pcr Based Assaymentioning

confidence: 99%

EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

Rustgi¹,

Bandopadhyay²,

Balyan³

et al. 2009

CZECH J GENET PLANT

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show abstract

“…starch synthase I (Li et al 1999a), starch synthase II (Li et al 1999b), starch synthase III (Li et al 2000), starch branching enzyme I , starch branching enzyme IIa (Rahman et al 2001) and isoamylase (Rahman et al 2003). Use of the cDNA sequences for D-enzyme identified the exon/intron structure of the partial genomic sequence from A. tauschii, and ten exons and nine introns were identified (Fig.…”

Section: The Characterisation Of the Wheat D-enzyme Gene From A Tausmentioning

confidence: 99%

Characterisation of disproportionating enzyme from wheat endosperm

Bresolin

Kosar-Hashemi

et al. 2005

Planta

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Disproportionating enzyme or D-enzyme (EC 2.4.1.25) is an alpha-1,4 glucanotransferase which catalyses cleavage and transfer reactions involving alpha-1,4 linked glucans altering (disproportionating) the chain length distribution of pools of oligosaccharides. While D-enzyme has been well characterised in some plants, e.g. potato and Arabidopsis, very little is known about its abundance and function in cereals which constitute the major source of starch worldwide. To address this we have investigated D-enzyme in wheat (Triticum aestivum). Two putative D-enzyme cDNA clones have been isolated from tissue-specific cDNA libraries. TaDPE1-e, from an endosperm cDNA library, encodes a putative polypeptide of 575 amino acid residues including a predicted transit peptide of 41 amino acids. The second cDNA clone, TaDPE1-l, from an Aegilops taushii leaf cDNA library, encodes a putative polypeptide of 579 amino acids including a predicted transit peptide of 45 amino acids. The mature polypeptides TaDPE1-e and TaDPE1-l were calculated to be 59 and 60 kDa, respectively, and had 96% identity. The putative polypeptides had significant identity with deduced D-enzyme sequences from corn and rice, and all the expected conserved residues were present. Protein analysis revealed that D-enzyme is present in the amyloplast of developing endosperm and in the germinating seeds. D-enzyme was partially purified from wheat endosperm and shown to exhibit disproportionating activity in vitro by cleaving maltotriose to produce glucose as well as being able to use maltoheptaose as the donor for the addition of glucans to the outer chains of glycogen and amylopectin.

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“…The melting temperature (Tm) should be considered one of the most important factors in order to achieve an allele-specific amplification [21]. The main difficulty to amplify the correct product of primers with GC-rich sequences was needed high temperatures to break up the strong secondary structure, formed by hydrogen bonds between the cytosine and guanine.…”

Section: Resultsmentioning

confidence: 99%

Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene

Zabala¹,

Gomez²,

Alvarez³

et al. 2017

OALib

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GWAS (genome-wide association studies) have associated Type 2 DiabetesMellitus with the single nucleotide polymorphism (SNPs) rs972283 (A/G) of KLF14 gene with in a global population. The most common techniques used to analyze SNPs are time consuming, multi-step process and require expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective T-ARMS PCR assay to genotype rs972283. However, the optimization step can be hardworking and laborious. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. In a first step, we design and validate two specific primer pair for T-ARMS PCR. Later, the amplification conditions were optimized for DNA concentration, annealing temperature, Taq DNA polymerase units and primers concentration. The last one was considered the main interference factor for a correct amplification and appropriate band intensity. Finally, the results obtained by T-ARMS PCR were concordant with sequencing. T-ARMS PCR assay developed in our laboratory for genotyping rs972283 (A/G) of KLF14 gene is time saving and cost-effective compared to the available methods used for SNP studies.

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Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS–PCR

Cited by 57 publications

References 19 publications

EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

Characterisation of disproportionating enzyme from wheat endosperm

Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene

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